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Published in final edited form as: Eur J Neurosci. 2025 Aug;62(4):e70232. doi: 10.1111/ejn.70232 Search in PMC Search in PubMed View in NLM Catalog Add to search Prelimbic Cortex Activity Predicts Anxiety-Like Behavior in the Elevated Plus Maze Marina A Smoak Marina A Smoak 1 Department of Biological Sciences, The University of Texas at El Paso, USA 3 Graduate Programs in Biosciences, The University of Texas at El Paso, USA Find articles by Marina A Smoak 1, 3 , Karla J Galvan Karla J Galvan 1 Department of Biological Sciences, The University of Texas at El Paso, USA 3 Graduate Programs in Biosciences, The University of Texas at El Paso, USA Find articles by Karla J Galvan 1, 3 , Daniel E Calvo Daniel E Calvo 2 Department of Psychology, The University of Texas at El Paso, USA 4 Graduate Programs in Psychology, The University of Texas at El Paso, USA Find articles by Daniel E Calvo 2, 4 , Rosalie E Powers Rosalie E Powers 1 Department of Biological Sciences, The University of Texas at El Paso, USA Find articles by Rosalie E Powers 1 , Travis M Moschak Travis M Moschak 1 Department of Biological Sciences, The University of Texas at El Paso, USA Find articles by Travis M Moschak 1 Author information Copyright and License information 1 Department of Biological Sciences, The University of Texas at El Paso, USA 2 Department of Psychology, The University of Texas at El Paso, USA 3 Graduate Programs in Biosciences, The University of Texas at El Paso, USA 4 Graduate Programs in Psychology, The University of Texas at El Paso, USA Author Contributions Marina A. Smoak : Data Curation, Formal Analysis, Visualization, Writing-original draft, Writing-review and editing; Karla J. Galvan : Investigation, Project Administration, Visualization, Writing-original draft; Daniel E. Calvo : Investigation, Writing-original draft; Rosalie E. Powers : Investigation, Visualization, Writing-original draft; Travis M. Moschak : Conceptualization, Formal analysis, Funding acquisition, Supervision, Writing-review and editing. ✉ Corresponding Authors: [email protected] (Travis M. Moschak), [email protected] (Marina A. Smoak), Present address: Department of Biological Sciences, The University of Texas at El Paso, 500 W University Ave, El Paso, TX 79968 PMC Copyright notice PMCID: PMC13082358 NIHMSID: NIHMS2103545 PMID: 41979977 The publisher's version of this article is available at Eur J Neurosci Previous version available: This article is based on a previously available preprint posted on bioRxiv on December 27, 2024: " Prelimbic Cortex Activity Predicts Anxiety-Like Behavior in the Elevated Plus Maze ". Abstract The medial prefrontal cortex plays a critical role in emotional regulation, and its dysregulation is linked to anxiety disorders. In particular, the prelimbic cortex of the medial prefrontal cortex is thought to modulate anxiety-related behaviors, though its precise role remains debated. Here, we used endoscopic in vivo calcium imaging to assess prelimbic neuronal activity in male and female Sprague-Dawley rats performing in the Elevated Plus Maze, a widely used task to measure anxiety-like behavior. We found that animals that spent less time in the open arms exhibited higher prelimbic activity in the open arms, suggesting that heightened prelimbic activity may reflect greater anxiety or increased avoidance behavior. These results suggest that the prelimbic cortex may play a role in regulating the emotional response to anxiety-provoking situations, potentially influencing the tolerance for exposure to threatening environments. Keywords: anxiety, prelimbic cortex, mPFC (medial prefrontal cortex), calcium imaging, GCaMP6s, rats Graphical Abstract Heightened activity in the prelimbic cortex of the medial prefrontal cortex correlates with increased anxiety-like behavior in rats. Using in vivo calcium imaging, we found that rats with greater prelimbic activity in the open arms of the Elevated Plus Maze spent less time in these exposed areas, suggesting a role for the prelimbic cortex in regulating emotional responses to anxiety-provoking situations. 1. Introduction The prefrontal cortex (PFC) integrates corticolimbic inputs to evaluate emotional significance and guide adaptive behavior ( McLaughlin et al., 2014 ). Variations in PFC activity are linked to reported anxiety levels ( Simpson et al., 2001 ), and dysregulation of PFC activity is associated with mental disorders such as generalized anxiety disorder (GAD) ( Cha et al., 2014 ), post-traumatic stress disorder (PTSD) ( Shin et al., 2005 ), and social anxiety ( Stein et al., 2002 ). Given these findings, it is essential to isolate and evaluate the activity of the PFC to clarify how normal emotional responses become dysregulated in anxiety-inducing conditions. The human PFC is a large region consisting of several subregions. Functional magnetic resonance imaging (fMRI) studies have demonstrated that participants diagnosed with GAD showed sustained activation of the anterior cingulate cortex [Brodmann area (BA) 32; ACC] ( Paulesu et al., 2010 ). In a recent fMRI study that evaluated individuals in an approach-avoidance task, high-anxiety participants relied on areas 24/25 (ACC) for emotional-action control, whereas their non-anxious peers did not ( Bramson et al., 2023 ). In rodents, the prelimbic cortex (PrL) of the medial prefrontal cortex (mPFC) is considered homologous to the ACC (areas 24/32) of non-human primates ( Heilbronner et al., 2016 ). Both inactivation ( Stern et al., 2010 ; Green et al., 2020 ) and activation ( Wang et al., 2015 ) of the PrL during elevated plus maze (EPM) testing has been shown to increase open arm exploration, suggesting an ambiguous and context-dependent role for the PrL in regulating anxiety-related behaviors in rodent models. To shed light on what might be driving these disparate findings following PrL manipulation, we investigated PrL neural activity in the EPM task. Specifically, this study assessed anxiety-like behaviors in male and female Sprague-Dawley rats using the EPM, a well-established paradigm for measuring anxiety phenotypes ( Hogg, 1996 ). During behavior, we used endoscopic in vivo Ca 2+ imaging to measure neuronal responses in the PrL. We found that increased PrL activity in the open arms was associated with less time spent in open arms. 2. Methods 2.1. Subjects and Surgery Female (n=5) and male (n=10) adult Sprague Dawley rats aged 8–10 weeks and weighing 200–300 grams were obtained from Envigo (St. Charles, MO). Animals were singly housed in a temperature-controlled room (21° C±1° C) under a 12-hour light/dark cycle (lights on at 8 PM) with food and water ad libitum. Upon arrival to the facility, animals were given 7 days to acclimate, followed with at least 7 days of handling by experimenters. To prevent damage to neural headgear by cage mates, rats were singly housed for the entirety of the experimental period, beginning immediately following surgical recovery and continuing through all behavioral testing. All experiments were conducted during the dark phase. All procedures were conducted following the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and in accordance with the University of Texas at El Paso Institutional Animal Care and Use Committee. Rats underwent stereotaxic surgery for intracerebral injection of the viral vector encoding the genetically coded calcium sensor GCaMP6s (pAAV.Syn.GCaMP6s.WPRE.SV40/100843-AAV5) and lens implantation in the prelimbic area (AP: +2.7; ML: +/− 0.6 DV: −3.6 for virus, −3.4 for lens). Infusions were counterbalanced for hemisphere (left or right). To secure the implantation, four stainless steel skull screws (Small Parts) were fastened to the skull, two rostrally and two caudally. In the PrL, a GRIN lens (1.0mm diameter, ~9.0mm length; Inscopix) was carefully implanted. After the lens was implanted and the skull thoroughly dried, fresh fast curing orthodontic acrylic resin powder and liquid (Lang Dental) were applied. Once completely hardened, a small protective boundary was created around the lens using dental cement. During surgery for intracerebral injection and lens implantation, animals were anesthetized with a cocktail of 100 mg/kg ketamine and 10 mg/kg xylazine and were given a one-week recovery period. Post-operative care included daily meloxicam and 10% enrofloxacin diluted in sterile saline. After one week of recovery, skin margin care was performed to ensure proper wound healing and to prevent infection. If fluorescence was detected in the brain after ~4–6 weeks, subjects then underwent installation of Miniscope baseplates where the animal was mildly anesthetized with isoflurane/oxygen vapor mixture and a 0.05 ml dose of 100 mg/kg ketamine and 10 mg/kg xylazine. Subsequently, animals underwent implantation of an intrajugular catheter with the port externalized at the back. For this procedure, animals were anesthetized with a cocktail of 100 mg/kg ketamine and 10 mg/kg xylazine. Post-operative care ensued with treatments of cefazolin, meloxicam, and daily flushing with 2% heparinized saline. Following recovery from these procedures and allowing time for virus expression (8–10 weeks), animals ran in behavioral tasks involving operant responding to sucrose pellets that were unrelated to the current study, followed by the elevated plus maze assessment mentioned below. 2.2. Elevated Plus Maze and Behavior A standard elevated plus maze constructed with black acrylic and equipped with infrared sensors at the entrance of each runway (Med Associates) was utilized to measure anxiety-like behaviors. The maze was placed in a silent and dimly lit room with an illumination of 100–200 lux, elevated 76.2 cm from the ground, and consisted of two open arms (49.53 × 11.43 cm) and two closed arms with no roof (49.53 × 11.43 × 43.18 cm) at right angles to each other, and an open square in the center (10.16 × 10.16 cm). During this task, rats were placed in the center square with their face pointing toward a closed arm of the elevated plus maze. The experimenter left the room, and subjects were left alone for 10 minutes. During this time, subjects were allowed to move freely within the apparatus. Infrared photobeam sensors placed along the walls of the maze determined the position of animals while in the elevated plus maze. Presence of the animal in a specific maze area (open arm, closed arm, or center) was defined by the interruption of at least two adjacent photobeams within that region, which also served to calculate the duration spent in each zone. 2.3. Endoscopic In Vivo Ca 2+ Imaging Imaging was recorded using software and hardware from the Miniscope system (UCLA Miniscope, Los Angeles, CA) with time-locked task events. Specifically, the Miniscope camera was attached to the implanted baseplate on the animal’s head and connected via the Miniscope data acquisition (DAQ) QT Software ( https://github.com/Aharoni-Lab/Miniscope-DAQ-QT-Software ). The DAQ system also included a transistor-transistor logic (TTL) connection to Med-PC (v.5), which controlled all behavioral systems and would trigger recordings captured using a high-powered Dell laptop. The program recorded neuronal activity for the entire 10 minutes of the task. The animal was removed from the maze and placed back in its home cage for 10 minutes of recording. This additional recording ensured enough neural activity was obtained for adequate detection of changes in fluorescence and subsequent identification of neurons but was not included in behavioral analysis. 2.4. Data Analysis Videos of calcium activity were obtained from Miniscope software, and time spent in the open arms versus the closed arms of the elevated plus maze was obtained through Med-PC. As described by Sullivan & Sdrulla (2022) , raw imaging files within the same field of view were concatenated into single, shorter videos containing timestamps using the Miniscope DAQ QT Software. Raw imaging files were assessed for quality before analysis and downsampled before being analyzed in CaImAn software ( Pnevmatikakis et al., 2016 ; Giovannucci et al., 2019 ). It is important to note that during miniscope recordings, animal movement occasionally causes mechanical shifts of the camera relative to the brain (e.g., from head movements or minor impacts). These shifts, detected as global motion of the field of view, were quantified by the built-in NoRMCorre camera motion correction algorithm implemented in CaImAn. Downsampled videos were median filtered and camera motion corrected in chronological order. Putative neurons were identified, restricted by a convolutional neural network classifier, and separated utilizing CaImAn’s nonnegative factorization method. This method simultaneously accounts for the spatial and temporal components of calcium signals. The activity of each putative neuron was estimated using the deconvolution algorithm within CaImAn. This algorithm contains parameters set to match the kinetics of GCaMP6s ( Chen et al., 2013 ). All putative neurons were manually inspected and artifactual or noisy neurons were excluded from further analysis. Once neural activity was detected in CaImAn, Matlab was used to align behavioral events that were captured by MedPC to neural activity. Extracted estimated activity trains were segmented according to whether they occurred while the animal was in the open arms, closed arms, or junction. For the quantification of mean neuronal activity for each identified ROI (neuron), we calculated the mean ΔF/F during open and closed arm epochs, respectively. To account for baseline differences in activity across neurons, these condition-specific means were then normalized by dividing by each ROI’s mean ΔF/F across the entire recording session (all arms). After normalization, we averaged across all ROIs within each animal to generate a single value per condition (open vs. closed) per subject. These values were then used to compute group-level means and statistical comparisons. We then calculated the relative neural activity per zone (open arms, closed arms, junction) over the total activity for the session as a measure of “Neural preference” (e.g., open arm activity / total activity). Furthermore, we classified individuals neurons as “Open-Preferring”, “Closed-Preferring”, or “No Preference” by comparing neural activity across open and closed arms entries using Wilcoxin rank sum tests. Two male animals spent less than 20s in the open arms and were thus excluded from the analysis as there was too little activity to accurately analyze. Statistical analysis was performed using IBM SPSS Statistics 29.0. Statistical significance level was set to α=0.05. Wilcoxon rank sum tests were used to compare sex, percent time spent in open arms, relative neural activity per arm, and percent open or closed preferring neurons. Median splits were performed on behavioral data such as number of open arm entries and percent time spent in open arms. Pearson correlations were used to compare neural activity to number of open arm entries and the percent time spent in open arms. To control for camera motion artifact, we also conducted partial correlations between neural activity and behavior while controlling for camera motion artifact. Finally, all data were collected by individuals blinded to the behavioral groups. With the exception of the final analysis (i.e. correlating neural activity with behavior), all data were analyzed by individuals blinded to the behavioral groups (e.g. extraction of neural activity, calculating mean neural activity per zone of the maze, etc.) 2.5. Histology Upon completion of the experiment, animals were euthanized via inhalation of a maximum amount of isoflurane/oxygen vapor mixture (5%) in a closed container. The animal was monitored and declared euthanized once signs that movement, respiration, and reflexes (extreme “toe pinch”) ceased. Tissue collection began after rapid decapitation following sacrifice where the brain was carefully removed, left overnight in 4% paraformaldehyde, and transferred to 30% sucrose the following day. Subsequently, the brains were set in optimal cutting temperature embedding medium (O.C.T., Fisher) and frozen on a tissue mount/specimen chuck in a cryostat (Leica CM3050 S Cryostat), sectioned coronally at 30 μm, and mounted on slides to verify lens placement. Verification was visualized via widefield fluorescence using a Zeiss LSM 900 with Airyscan 2 confocal microscope and placement was mapped onto an atlas (Paxinos and Watson, 2007). Data were only included if cells and lens placement were verified to be in the prelimbic cortex. 3. Results 3.1. PrL neural activity predicts time spent in open arms Animals were classified using a median split into groups with low or high percent time spent in the open arms and low or high number of open arms entries. Subjects in the Low group could be considered to have higher anxiety-like behavior and subjects in the High group could be considered to have lower anxiety-like behavior ( Fig. 1A ). Median splits were also considered for percent time spent in closed arms and percent time spent in the junction of the elevated plus maze. Groups were then further evaluated for differences in prelimbic activity. Data from an example subject can be seen in Fig. 1B , C . Recordings ranged between 5 and 158 neurons ( Fig. 1D ). Subjects who spent less time in the open arms had a significantly higher proportion of PrL activity in the open arms when compared to subjects who spent more time in the open arms ( Fig. 1E , Wilcoxon W = 25.00, Z = −2.242, p = 0.026, HI = 1.04 ± 0.03, LO = 1.23 ± 0.04). No differences in PrL activity were seen when comparing proportion of PrL activity in the closed arms ( Fig. 1F , Wilcoxon W = 33.00, Z = −0.961, p = 0.394, HI = 0.98 ± 0.01, LO = 0.96 ± 0.01) or proportion of PrL activity in the junction ( Fig. 1G , Wilcoxon W = 34.00, Z = 0.801, p = 0.485, HI = 1.00 ± 0.04, LO = 1.03 ± 0.06). When comparing all subjects, Pearson’s correlations revealed a relationship between the proportion of PrL activity in the open arms and the percent time spent in open arms (r = −0.56, p = 0.04; Fig. 1H ), where higher PrL activity was associated with more anxious behavior. We saw no relationships between the proportion of PrL activity in the closed arms and the percent time spent in the open arms (r = 0.29, p = 0.33; Fig. 1I ) and the proportion of PrL activity in the junction and the percent time spent in the junction (r = −0.15, p = 0.61). Taking camera motion into account did not change this relationship (partial correlations: Open arm activity: r = −0.632, p = 0.027; Closed arm activity: r = 0.337, p = 0.285; Junction activity: r = −0.187, p = 0.56). We then examined activity in individual neurons that statistically preferred either the open or closed arms. We found no difference between High and Low animals in the percent of all neurons that statistically preferred the Open (Wilcoxon W = 38.00, Z = −0.166, p = 0.937; Fig. 2A ) or Closed arms (Wilcoxon W = 36.00, Z = −0.481, p = 0.699; Fig. 2B ). Next, we looked only at the subset of neurons that had a statistical preference (excluding all neurons that did not). For each animal, we calculated the relative percent of these neurons that preferred the open arm over the closed arm. We found that the proportion of neurons per rat that preferred open over closed did not differ by group (Wilcoxon W = 25, Z = −0.541, p = 0.69; Fig. 2C ), nor did a correlation of this neural preference predict percent time in the open arms (r = −0.186, p = 0.586; Fig. 2D ). Taking camera motion into account did not change this relationship (partial correlation: r = −0.369, p = 0.264). Figure 1. Open in a new tab Behavioral and neural analysis of anxiety-like behaviors. (A) Distribution of percent time spent in open arms, closed arms, and junction for all subjects where the median for each is shown as a threshold. (B) Miniscope field of view and regions of interest (ROIs) as extracted by CaImAn. (C) Calcium traces of example neurons. (D) Number of neurons analyzed per subject. (E) Mean neuronal activity for the percent time spent in open arms (W = 25.00, Z = −2.242, p = 0.026, Wilcoxon rank sum test, HI = 1.0 4± 0.03, LO = 1.23 ± 0.04), (F) closed arms (W = 33.00, Z = −0.961, p = 0.394, Wilcoxon rank sum test, HI = 0.98 ± 0.01, LO = 0.96 ± 0.01), and (G) junction (W = 34.00, Z = 0.801, p = 0.485, Wilcoxon rank sum test, HI = 1.00 ± 0.04, LO = 1.03 ± 0.06) The data are presented as mean ± SEM. *p ≤ 0.05; **p ≤ 0.01. Pearson’s correlations for percent time spent in open arms and mean neuronal activity for (H) open arms, (I) closed arms, and (J) junction. Figure 2. Open in a new tab Analysis of Neural Preference & Histology. Percent time spent in open arms and percent neurons preferring (A) open arms (W = 38.00, Z = −0.166, p = 0.937, Wilcoxon rank sum test, HI = 2.72 ± 0.93, LO = 3.48 ± 1.87), and (B) closed arms (W = 33.00, Z = −0.961, p = 0.394, Wilcoxon rank sum test, HI = 0.98 ± 0.01, LO = 0.96 ± 0.01), and (C) percent phasic neurons preferring open arms (W = 25.00, Z = −0.551, p = 0.69, Wilcoxon rank sum test, HI = 23.63 ± 11.26, LO = 30.83 ± 14.52) The data are presented as mean ± SEM. *p ≤ 0.05; **p ≤ 0.01. (D) Pearson’s correlation for percent time spent in open arms and percent phasic neurons preferring open arms. (E) Example of fluorescent image of coronal section used to neuroanatomically verify lens placement and (F) all placements mapped onto templates of a rat brain atlas (Paxinos & Watson, 2006). 4. Discussion Overall, the observed increase in PrL activity in the open arms among animals with lower open arm exploration suggests a potential neural correlate of heightened anxiety-like behavior. Given the open arms represent an anxiogenic context, increased PrL firing may reflect an overactive top-down appraisal of threat, consistent with the PrL’s established role in attention and cognitive control during aversive experiences. Since the amount of time spent in the open arms of the EPM may reflect the subject’s level of anxiety or willingness to remain in an exposed, potentially threatening environment, PrL neural activity could be linked to the animal’s tolerance of/coping with anxiety ( Aliczki et al., 2016 ), or greater exploration despite fear ( Casanova et al., 2024 ; Marin-Blasco et al., 2024 ) in this paradigm. Our results also indicated that there was a lack of significant group differences in PrL activity within the closed arms and junction. This suggests that PrL hyperactivity is context-dependent, selectively heightened in threatening environments. Our findings reinforce many previous preclinical outcomes. For example, temporary PrL inactivation during EPM testing increased open arm exploration ( Stern et al., 2010 ; Green et al., 2020 ). Additionally, lesions of the entire mPFC (including the PrL) yielded a higher percentage of open arm entries and time spent in open arms ( Lacroix et al., 2000 ). Furthermore, effects of dorsal and ventral mPFC infusions of the benzodiazepine midazolam produced anxiolytic effects as determined by greater open arm entries and percentage of time in open arms (Shah et al., 2004). Finally, optogenetic suppression of the PrL-BLA (basolateral amygdala) pathway in chronic pain mice increased time spent in open arms ( Gao et al., 2023 ). However, these effects may depend on neural subtype and receptor. Notably, activation of mPFC β-adrenoceptors in excitatory neurons was anxiogenic ( Lei et al., 2022 ), and intra PrL injection of a selective dopamine D4 receptor antagonist increased time spent in the open arms ( Vergara et al., 2017 ), while activation of either Ca 2+ /calmodulin-dependent protein kinase α (CamKIIα)-positive excitatory neurons in the PrL ( Pati et al., 2018 ) or Drd1 expressing neurons in the mPFC produced anxiolytic responses ( Hare et al., 2019 ). Furthermore, some studies have found null or even opposing effects of PrL manipulation on anxiety-like behavior. Stimulation of the PrL had no effect on EPM behavior in one study ( Shimizu et al., 2018 ), while optogenetically activating the contralateral PrL to an inflamed hind paw produced anxiolytic effects ( Wang et al., 2015 ). Furthermore, reversible inactivation of bilateral ventral portions of the mPFC (including the PrL) enhanced anxiety during EPM testing as determined by decreased number open arm entries ( Lisboa et al., 2010 ). Nonetheless, our findings support the bulk of studies manipulating the PrL that suggest that heightened activity in the PrL is associated with heightened anxiety-like behavior ( Lacroix et al., 2000 ; Stern et al., 2010 ; Green et al., 2020 ; Gao et al., 2023 ). Few studies have assessed PrL neural activity during the EPM. In one, recording of multiunit activity via wireless telemetry revealed no change in the firing rate of PrL neurons during open arm entries, indicating that PrL neurons may not play a role in overcoming anxiety-like behaviors ( Shimizu et al., 2018 ). While our results did not replicate these findings, other studies have provided evidence that there are dynamic changes in PrL activity between safe and aversive locations in the EPM. Lu et al., (2018) reported strengthened theta oscillations in PrL functional connectivity and increased information transfer efficiency while in the closed arms of the EPM. This further supports that anxiety-induced changes in neural activity occur in the PrL and enhanced connectivity may contribute to the inhibition of exploratory behaviors, which the PrL is known to encode ( Ahmadlou et al., 2021 ; Brockway et al., 2023 ). In addition to rodent work, clinical studies suggest that individuals with anxiety show increased ACC activation, especially when tasked with emotional regulation or threat anticipation ( Amir et al., 2005 ; Simmons et al., 2008 ; Paulesu et al., 2010 ; Maier et al., 2012 ; Fullana et al., 2016 ; Bramson et al., 2023 ; Buehler et al., 2024 ), although activation patterns vary depending on the specific type of anxiety disorder or task ( Burkhouse et al., 2018 ). These clinical findings, which highlight increased ACC activation in anxiety contexts, align with our preclinical observations and those of others, suggesting that similar neural circuits, particularly within the mPFC, may play a central role in the regulation of anxiety across species. There are several final considerations when contextualizing our findings. While overall PrL activity in the open arms was predictive of anxiety-like behavior, neural preference at the single-cell level did not differentiate high- and low-anxiety animals. This null finding may be partly due to the low proportion of phasic cells across subjects; notably, when restricting the analysis to animals with at least one phasic cell, we observed a strong negative correlation in the expected direction (r = −0.86, p = 0.062; n = 5), suggesting that greater phasic engagement may be more tightly linked to individual anxiety levels. Although our neuron-level analyses did not show a clear relationship between cell preference and anxiety-like behavior, the overall pattern of PrL activity suggests a more complex neural dynamic. Specifically, while our statistical analyses indicated that some neurons did not exhibit significant modulation between the open and closed arms, the population-level data ( Fig. 1 ) reveal an overall pattern of activity that varies with arm type. This observation suggests that PrL neurons may display mixed or overlapping tuning properties—engaging during both open and closed arms but with distinct activity profiles that likely encode complex features of the environment. One alternative explanation for increased PrL activity, particularly in the open arms, may be related to top-down motor inhibition rather than anxiety per se. While we interpret increased PrL activity in the open arms as related to anxiety-like behavior, an alternative explanation is that this activity reflects top-down inhibition of impulsive motor responses. The PrL has been widely implicated in inhibitory control and executive function, including suppression of unwanted actions. Future studies combining anxiety and impulsivity paradigms could clarify whether PrL activity in this context reflects affective or cognitive control processes. Finally, because rats were singly housed throughout the duration of behavioral testing, it is important to consider the potential influence of social isolation on anxiety-like behavior. Isolation has been shown to alter activity in stress-related neural circuits, including the prefrontal cortex, and can increase anxiety-like responses in tasks such as the elevated plus maze ( Yamamuro et al., 2018 ; Manouze et al., 2019 ; Yamamuro et al., 2020 ). While single housing was necessary to protect neural implants and ensure data quality, the effects of social isolation on neural activity and behavior should be considered when interpreting these findings. Finally, a limitation of our study is the absence of video-based tracking of animal locomotion during elevated plus maze testing. As such, we were unable to directly assess whether gross physical movement contributes to variance in calcium signals independently of arm occupancy. Future studies incorporating synchronized behavioral video tracking could address this potential confound more comprehensively. Conclusion We found that subjects with higher anxiety-like behavior exhibited increased PrL activity in the open arms of the EPM. These results validate previous findings and strengthen the reliability of the prelimbic cortex’s role in anxiety across different methodologies and experimental conditions. Future studies could focus on the dynamics of this relationship by incorporating real-time tracking of activity in PrL projections implicated in EPM. One pathway of interest is the PrL’s projection to the basolateral amygdala, as optogenetic suppression of PrL neurons projecting to the basolateral amygdala increased the time spent in open arms ( Gao et al., 2023 ). The ventral hippocampus is an alternative target of interest that projects to the PrL, since in the retrieval of fear extinction memory in rodents, the primary input to active neurons in the PrL came from the ventral hippocampus ( Szadzinska et al., 2021 ). Finally, it would be valuable to examine how individual differences, such as genetic factors within the PrL ( Chen et al., 2017 ) or it’s inputs ( Hallock et al., 2020 ), or prior stress exposure (Corcoran et al., 2007; Quiñones-Laracuente et al., 2021 ; Smiley et al., 2021 ), modulate PrL activity or connectivity and anxiety responses, which may help tailor more personalized approaches to anxiety treatment. Acknowledgements This work was supported by National Institute on Drug Abuse (NIDA) grant DA045764 and National Institute of Health (NIH) grant U54MD007592-28 awarded to TMM and the Dr. Keelung Hong Graduate Research Fellowship awarded to MAS. The authors would also like to thank Peter A. Fogel for the valuable discussions. Abbreviations PFC prefrontal cortex GAD generalized anxiety disorder PTSD post-traumatic stress disorder fMRI functional magnetic resonance imaging ACC anterior cingulate cortex mPFC medial prefrontal cortex GRIN gradient-index micro lens BA Brodmann area PrL prelimbic cortex EPM elevated plus maze DAQ miniscope data acquisition TTL transistor-transistor logic BLA basolateral amygdala Footnotes Competing Interests The authors declare no competing financial interests. Data Accessibility The data presented in this report are available in the Harvard Dataverse repository ( Moschak, 2024 ; https://doi.org/10.7910/DVN/RYAHYV ). References Ahmadlou M, Houba JHW, Vierbergen J.F.M.van, Giannouli M, Gimenez G-A, Weeghel C. van, Darbanfouladi M, Shirazi MY, Dziubek, Kacem M., Winter F. de, & Heimel JA. (2021) A cell type–specific cortico-subcortical brain circuit for investigatory and novelty-seeking behavior. Science, 372. 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